A variety of different therapeutic concepts have been used for the treatment of cancer patients. In the past years clinical trials have been performed with monoclonal antibodies, which recognize specifically or preferentially cell surface molecules expressed on malignant cells. Aim of this approach is the induction of antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC) to eliminate tumor cells. A second approach is the cytokine-mediated activation of an immune response. The cytokine-induced anti-tumor activity can be mediated by:    1) a direct cytotoxic/cytostatic effect of the cytokine on tumor growth    2) tumor-antigen-nonspecific mechanisms such as LAK activity or monocyte/macrophage mediated cytotoxicity    3) tumor-antigen specific immune responses mediated by CD4 and CD8-positive T cells. In this situation a systemic immunity against the tumor has been observed in animal models.
Unfortunately, systemic application of cytokines such as IL-2 or TNFα is hampered by their toxicity (Rubin, Cancer Invest. 11:460–472, 1990; Balkwill, Nature 361:206–207, 1993). To ensure a sufficient cytokine concentration at the tumor site rather high doses have to be administered and the maximumly tolerable dose is below the dose required for efficacy. Therefore, the negative effects of cytokines are mainly a consequence of the systemic application, but their clinical relevance in tumor therapy is undoubted. In animal models it was demonstrated that in situ presence of the cytokine either by intratumoral injection or by secretion of transfected tumor cells may lead to tumor regression (Hock et al. PNAS 90:2774–2778, 1993; Colombo et al. Cancer Res. 52:4853–4857, 1992; McBride et al., Cancer Res. 52:3931–3937, 1992; Tepper et al., Science 257:548–551,1992; Mullen et al., Cancer Res. 52:6020–6024, 1992; Blankenstein et al., J. Exp. Med 173:1047–1052,1991; Gansbacher et al., J. Exp. Med. 172:1217–1224, 1990). In these systems, cytokines do not impair tumor proliferation, but are capable of activating a rapid and potent anti-tumor reaction. Therefore, the physical combination of an effector molecule and a targeting element represents a means of reducing the peripheral presence and enhance the intratumoral availability of the biologically active ligand. Furthermore, single tumor cells or micro-metastases can also be targeted by these molecules.
The biologically active ligand for an antibody-directed targeting should induce the destruction of the target cell either directly or through creating an environment lethal to the target cell. The biologically active ligand can be a cytokine such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-13, IFNs, TNFα or CSFs. These cytokines have been shown to elicit anti-tumor effects either directly or by activating host defense mechanisms (Mire-Sluis, TIBTECH 11:74–77,1993; Colombo et al. Cancer Res. 52:4853–4857, 1992; Thomas & Balkwill, Pharmac. Ther. 52:307–330, 1991).
For instance, IL-2 is considered the central mediator of the immune response. II-2 has been shown to stimulated the proliferation of T cells and NK cells and to induce lymphokine-activated killer cells-(LAK). Tumor-infiltrating T lymphocytes proliferate in response to IL-2. Furthermore, II-2 enhances the cytotoxicity of T cells and monocytes. In addition, IL-2 induces a cytokine cascade secreted by T cells, NK cells and monocytes which further potentiate the immune response.
TNFα has found a wide application in tumor therapy, mainly due to its direct cytotoxicity for certain tumor cells and the induction of hemorrhagic regression of tumors. In addition, TNFα potentiates the immune reponse: it is a costimulant of T cell proliferation, it induces expression of MHC class I and class II antigens and TNFα, IFN and IL-1 secretion by macrophages. IL-4 was initially described as a B cell growth factor. In further studies IL-4 was shown to stimulate antigen-specific cytotoxic T cells and to act specifically on T cell-like LAK cells rather than NK cell like LAK cells. IL-4 inhibits the growth of human melanoma cells enhances their MHC class I and II expression. The induction of macrophage mediated anti-tumor effects by IL-4 is still controversial.
IL-7 is a growth factor for pre-B cells as well as for human peripheral CD4 and CD8 positive T cells. IL-7 directly augments the cytotoxicity of the CD8-positive T cell subpopulation. In addition IL-7 induces the production of IL-1, IL-6 and TNFα by peripheral monocytes. In vitro, the tumoricidal activity of monocytes/macrophages can be stimulated by IL-7, possibly mediated by cytokines, such as TNFα.
Epidermal growth factor (EGF) is a polypeptide hormone which is mitogenic for epidermal and epithelial cells. When EGF interacts with sensitive cells, it binds to membrane receptors (EGFR). The EGFR is a transmembrane glycoprotein of about 170 kD, and is a gene product of the c-erb-B proto-oncogene.
The murine monoclonal antibody MAb 425 was raised against the human A431 carcinoma cell line (ATCC CRL 1555) and found to bind to a polypeptide epitope on the external domain of the EGFR. It was found to inhibit the binding of EGF and to mediate tumor cytotoxicity in vitro and to suppress tumor cell growth of epidermal and colorectal carcinoma-derived cell lines in vitro (Rodeck et al., 1987, Cancer Res. 47, 3692). Humanized and chimeric versions of MAb 425 are known from WO 92/15683.
Antibody-cytokine immunoconjugates in various combinations for the directed delivery of active proteins have been described previously. IL-2 was combined with the human carcinoma-specific antibody L6 (Fell et al., 1991, J. Immunol. 146:2446–2452, EP-OS-0439095), or with an anti-ganglioside GD2 antibody (Gillies et al., 1992, PNAS 89:1428–1432, WO 92/08495). Immunoconjugates comprising of an anti-dansyl antibody and IGF1 have been generated for the directed delivery of hormones (Shin & Morrison, 1990, PNAS 87:5322–5326, WO 91/14438).
Thus, it was object of the invention, to create antibodies or fragments thereof comprising (1) an epitope directed to an EGFR antigen on the surface of a tumor cell and (2) a biologically active ligand having a high potential for induction of cytotoxicity, and, therefore, intensifying the anti-tumor effect in situ.